Many diagnostic tests are designed for use in well-equipped clinical labs. There are fundamental limitations to using and producing these tests in a resource-limited environment, especially those in developing nations where laboratory equipment and diagnostic tools are scarce or non-existent. The requirements for most basic diagnostic tests are i) liquid handling; ii) measurement of liquids and reagents; iii) mixing of liquids and reagents; iv) incubation of reagents; v) a binding assay that senses the presence of the analyte to be detected; vi) amplification of the detection signal; and vii) the readout and analysis of the detected signal. The foregoing requirements are difficult if not impossible to meet in a developing country, especially those areas with limited resources, for example refugee camps. It is precisely in such resource limited environments where a simple, low cost detection device and method are required. Additional requirements for diagnostic tests in developing countries must take into consideration the following: viii) the use of low-cost, on-site components; ix) the ability to produce devices without advanced infrastructure i.e., in the absence of roll-to-roll, robotic, conveyer belt and/or injection molding processes etc.; x) simple storage in the absence of refrigeration and/or incubation of samples; and xi) a lack of electricity to power diagnostic equipment.
Most if not all diagnostic assays satisfying criteria i) to vii) require production in advanced settings. Examples include devices that require high-resolution microfluidic channels, electronic components, and reagents that require storage at low temperature. However, such devices and systems do not satisfy requirements viii) to xi), and are not suitable for production or use in low-resource environments or in the field.
Phage display is a well know technique used in the analysis, display and production of protein antigens, especially human proteins of interest. Phage display is a process during which the phage, a bacterial virus, is made to expose or “display” different peptides or proteins including human antibodies on its surface. Through genetic engineering, peptides or proteins of interest are attached individually to a phage cell surface protein molecule (usually Gene III protein, g3p). In such a phage population (phage library), each phage carries a gene for a different peptide or protein—g3p fusion and exposes it on its surface. Through a variety of selection procedures, phages that “display” binders to specific target molecules of interest can be identified and isolated. The phage display technique is very useful in discovery and development of pharmaceutical and/or diagnostic products. In phage display the entire phage binds and can be eluted from an immobilized target molecule. Since the phage remains infective it can inject its DNA into bacterial cells and is amplified. The main limitation of phage display, however, is the occurrence of non-specific adsorption of phages during the binding stage, which necessitates enrichment over several rounds and individually tailored washing and elution conditions. Detection of such enrichment requires sequencing of the phage genome to detect emergence of the specific binding motif. Accordingly, these requirements make phage display technology impractical for diagnostic detection in low-resource environments.
Selectively infective phage (SIP) technology is another technique which can be used to screen large libraries of proteins or other oligo- or polypeptides to select antibodies having high affinity for a target antigen. This technology is described in U.S. Pat. No. 5,514,548. SIP technology is related to phage display technology in that it uses filamentous phages, where a protein of interest is fused to a phage coat protein and, thus, displayed on the outer surface of the phage, while its genetic information is contained in the phage DNA. In contrast to phage display, the phage particles in SIP technology are rendered non-invective by disconnecting the N-terminal domains (N1 and N2) of the phage g3p coat protein which are involved in docking to and penetrating a bacterial cell from the C-terminal domain (CT), which caps the end of the phage. The N- and C-terminal domains are then each fused to one of the interacting partners being studied. One partner is displayed on the phage surface associated with the CT, while the other is genetically fused or chemically coupled to the NT(s), thus making a separate adapter molecule. Only when the specific protein-ligand interaction occurs between the partners is the g3p reconstituted so that the phage particle regains its infectivity, and the genetic information of a successful binder is propagated. The main advantages of SIP technology over phage display are i) that it can be carried out in a continuous manner, which mimics the in vivo system of clonal selection used by the immune system for antibody optimization; ii) no solid-phase interaction with a support is required, reducing the occurrence of non-specific interactions and eliminating the need for elution; iii) reduction of background infectivity; and iv) high sensitivity as single binding events are detectable. However, for SIP technology to achieve these advantages, the genome of SIP must be non-infective rendering the progeny of SIP also non-infective. Amplification of signal in SIP, therefore is limited. SIP also necessitates genetic fusion or chemical covalent attachment of the binding partners to the coat proteins of phage. Binding of molecules that are not covalently attached to phage proteins cannot be detected by SIP technology. Extension of SIP to detection of soluble analytes, as it is necessary for diagnostic applications, is not obvious. These limitations make SIP technology unsuitable as a diagnostic technique.
Three-dimensional cellular arrays have been well described in WO 2009/120963 (Derda et al. (1)) and in “Paper-Supported 3D Cell Culture for Tissue-Based Bioassays” Proc. Natl. Acad. Sci. 2009, 106(44), 18457-18462, (Derda et al. (2)). The three-dimensional cellular arrays include a porous hydrophilic substrate with a number of porous regions bounded at least in part by a liquid impervious boundary and a hydrogel comprising cells, wherein the hydrogel is embedded within the porous regions of the device. The substrate may be constructed from paper, nitrocellulose, cellulose acetate, cloth, or porous polymer film.
These three-dimensional cellular arrays have been used to characterize various properties of cells grown within the arrays using well known cellular assays, including apoptosis, cell proliferation, cell cycle and gene expression. The three-dimensional cellular arrays may also be used to assay any one or more synthetic, naturally occurring, or a combination of synthetic and natural test agent, for example urine, blood, tears, sweat, or saliva, wherein a change in cellular function in the presence of the one or more test agents indicates the one or more test agents modify cellular function. However, in order to mimic a three-dimensional environment most of the cellular arrays disclosed in WO 2009/120963 require a hydrogel or hydrogel precursor be embedded within the porous regions. The viability of Pseudomonas aeruginosa strain PA 14 cells grown in a stack of 200 micron chromatography paper was investigated as set out in Example 9 of WO 2009/120963. The result was that a steady decrease in the number of both live and dead bacteria in the middle of the array was observed. This was attributed to competing rates of oxygen diffusion and oxygen consumption by the bacteria. All examples and claim presented in the WO 2009/120963 use paper-based 3D culture as a part of another sterile culture, such as submersions in sterile media incubated in a sterile container, housed inside a humidity and temperature controlled aseptic environment with humidity and temperature control. It is non-obvious how to extend this invention to create an autonomous setup that requires no secondary sterile container and no aseptic incubator. Accordingly, the difficulty in maintaining cell viability within the three-dimensional cellular arrays as well as the need for advanced incubation and imaging equipment to monitor biological activity within these types of 3-D arrays makes them inadequate for the purposes of a simple diagnostic assay.
Given the foregoing, there remains a need for a diagnostic device and method that can be easily constructed from simple components, on-site in low-resource environments.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.